Multiple biochemical pathways are available to substrates after entering the cytoplasm of the myocardium. These substrates may be oxidized, incorporated into intracellular storage sites, or converted to TCA cycle intermediates without net oxidation. The latter pathways which replenish the concentration of TCA cycle intermediates are collectively termed the anaplerotic reactions in this proposal. Those pathways, particularly pyruvate carboxylation, are active in the mammalian heart and respond to changes in O2 delivery, available substrate, and significant changes in cardiac work. However, the quantitative activity of the anaplerotic reactions has not been investigated systematically. Furthermore, there is considerable controversy over which substrate(s) is (are) oxidized by the heart under various physiological conditions when more than one is available. We propose to use 13C NMR to establish the precise biochemical fate of 13C-enriched substrates as they are metabolized by perfused hearts and in vivo hearts under normoxic, hypoxic, and increased work-load conditions. We present a method to the relative anaplerotic and oxidative activities and the fractional enrichment of molecules entering these pathways from a single NMR spectrum obtained under steady-state perfusion conditions. Metabolic data will he obtained for four different physiological substrates, and all combinations thereof in control rats, in fasted rats, and in hypertensive rats. These data will define the relative importance of anaplerotic flux in the heart during these varied physiological conditions and provide quantitative evidence of the substrate(s) selected for oxidation in response to these interventions. 13C NMR offers a great deal of potential for examining metabolism in vivo and this systematic study will allow us to establish a basis for and the limitations of 13C spectroscopy to probe intermediary metabolism in animals and in humans.